In vitro nucleic acid amplification techniques are now commonly used for synthesizing, and perhaps detecting vanishingly small quantities of a nucleic acid target. These techniques conventionally employ one or more oligonucleotide primers and a nucleic acid-polymerizing enzyme to synthesize copies of one or both strands of a nucleic acid template. Many different methods have been used for preparing biological samples in advance of the amplification procedure.
Multiplexed assays, which are capable of amplifying any of a plurality of different nucleic acid targets from a test sample in a single reaction, present special design challenges. For example, the targets amplifiable in a multiplex assay may be RNA targets, DNA targets, or even a combination of RNA and DNA targets. One challenge arises from the fact that RNA and DNA nucleic acids exhibit different chemical stabilities. Another challenge arises from a common desire to detect, with maximum sensitivity, any of a variety of related subtypes of a single target species. Even when subtype-specific primers are used in the reactions, it can be difficult to achieve substantially similar detection sensitivity for different subtypes of a single type of organism.
Accordingly, there is a need for a general technique which can enhance detectability of particular targets in nucleic acid amplification reactions. There is a further need for enhancing detectability of one or more targets in multiplex amplification reactions without substantially sacrificing detectability of other targets in the same reaction. The present invention addresses these needs.
Indeed, the invention disclosed herein provides a convenient method for preparing biological samples to be tested for the presence of nucleic acid targets using in vitro nucleic acid amplification. This method advantageously provides reliable results with a variety of nucleic acid-containing biological samples, while dramatically improving detectability of certain nucleic acid targets.